Protein tyrosine kinases are intimately involved in T cell signaling via the TCR/CD3 molecular complex. The role of heterotrimeric G proteins in this process is not clearly established. In view of recent evidence that the tyrosine kinase- and G protein-regulated pathway "cross-talk" to each other, we are proposing to study the involvement of G proteins in T cell signal transduction. We first plan to study the physical association between G proteins and TCR/CD3. We'll utilize chimeras and mutants of the intracytoplasmic trail of the CD3xi chain in an attempt to define the critical sites involved in the physical association, and specific peptides representing sites on Galpha subunits to determine whether they can interfere with the association. Co-immunoprecipitation and western blotting analyses with specific antibodies, and CD3xi- GST fusion proteins will be the primary tolls utilized in order to address these issues. The analysis will be extended to human thymocytes, peripheral T cells, and mouse splenic cells in order to better establish the biologic significance of the physical associations. Furthermore, G proteins "function-deficient" mutants will be utilized to further explore the significance of the physical associations. We will next utilize co-immunoprecipitation and western blotting analyses with anti-tyrosine kinase antibodies, as well as specific peptide competition in order to investigate the association of G proteins with specific protein tyrosine kinases. G protein "function-deficient" mutants will be utilized to assess the functional relevance of the physical associations. Further, we will assess the involvement of G proteins in the stimulation of ras and the subsequent activation of the MAP-kinase cascade, by studying the interaction of ras-GTP exchange factors with G proteins and the effects of G protein "function-deficient" mutants on this interaction. The biological significance of the above findings will be assessed by determining effects on late events such as raf and MAP-kinase activation, as well as IL2 production, and CD69 expression. The already established systems, the available technical expertise, the existing data, and the feasibility of the proposed experiments render this proposal straightforward, and likely to yield new and important information. In addition, our findings will provide important insights for future experimentation.